Recombinant human secreted DDRGK1-Fc and application thereof

Abstract

This invention relates to a recombinant human secretory DDRGK1-Fc and its applications. Through experimental verification, the invention synthesizes recombinant human secretory DDRGK1-Fc, which has good bone-promoting function and is applied to the preparation of drugs for treating bone and joint-related diseases, promoting cartilage formation, and promoting intervertebral disc formation. This opens up new ideas for the treatment of bone and joint diseases and has obtained relatively solid scientific evidence.

Description

Technical Field

[0001] This invention belongs to the field of bone and joint disease treatment, and specifically relates to a recombinant human secretory DDRGK1-Fc and its application. Background Technology

[0002] Currently, the treatment for bone-related diseases mainly involves medication or surgery. However, the excessive trauma of surgery often leads to delayed healing or nonunion of fractures, and there is often a lack of effective osteogenesis-promoting programs for these conditions. For osteoporosis, treatment often relies solely on medication to increase bone mass, as there is currently no effective surgical cure.

[0003] DDRGK1 consists of 314 amino acids, of which 1-28 are signal peptides that determine its localization in the endoplasmic reticulum (ER) or its exocrine location. Studies have found that DDRGK1 and UFL1 are mainly located in the ER, and that DDRGK1 and UFL1 can serve as substrates for Ufm1, thereby participating in ubiquitination modification. In this process, the lysine residue at position 267 of the DDRGK1 protein is crucial for its binding to UFM1 (A Novel Type of E3 Ligase for the Ufm1 Conjugation System, 2010). High expression of ubiquitin-related proteins, including UFM1, UFL1, DDRGK1, and CDK5RAP3, has been found in pancreatic Langerhans cells and other secretory cells. UFM1's endoplasmic reticulum (ER) localization depends on DDRGK1 binding. Silencing UFM1 or DDRGK1 has been shown to induce ER stress in pancreatic B cells and promote apoptosis. Therefore, UFM1-DDRGK1-mediated ubiquitinization is involved in regulating ER stress-induced apoptosis (Ubiquitin fold modifier 1 (UFM1) and its target UFBP1 protect pancreatic beta cells from ER stress-induced apoptosis, 2011). DDRGK1 directly binds to IκBα, inhibiting its phosphorylation and ubiquitination degradation, thereby enhancing IκBα stability. DDRGK1 knockout inhibits the NF-κB signaling pathway (DDRGK1 regulates NF-κB activity by modulating IκBα stability, 2013).

[0004] Fc is equivalent to the CH2 and CH3 functional regions of IgG. It is named for its crystallizable chemical characteristic. It has no antigen-binding activity, but it has functions such as fixing complement and binding Fc receptors. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a recombinant human secretory DDRGK1-Fc and its application. Through experimental demonstration, this invention synthesizes recombinant human secretory DDRGK1-Fc, which has good bone-promoting function and is applied to the preparation of drugs for treating bone and joint-related diseases, promoting cartilage formation and intervertebral disc formation. This opens up new ideas for the treatment of bone and joint diseases and has obtained relatively solid scientific evidence.

[0006] The present invention provides a recombinant human secretory DDRGK1-Fc, wherein the DDRGK1-Fc comprises a recombinant human secretory DDRGK1 that has been reprogrammed at 1-28 sites to change its location from cell membrane to secretory type, wherein the N-terminus of the DDRGK1 is connected to HA and the C-terminus of the DDRGK1 is connected to the Fc fragment.

[0007] Preferably, the amino acid sequence of the HA is: MKTIIALSYIFCLVFAGRA.

[0008] Preferably, the Fc fragment amino acid sequence is as follows:

[0009] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0010] The present invention also provides the application of recombinant human secretory DDRGK1-Fc in the preparation of drugs for treating bone and joint-related diseases.

[0011] Preferably, it is used in the preparation of drugs for treating osteoporosis, fractures or bone defects.

[0012] This invention also provides the application of recombinant human secretory DDRGK1-Fc in the preparation of drugs that promote cartilage formation.

[0013] This invention also provides the application of recombinant human secretory DDRGK1-Fc in the preparation of drugs that promote intervertebral disc formation.

[0014] The present invention also provides a drug comprising the above-described recombinant human secretory DDRGK1-Fc.

[0015] Preferably, the dosage form of the drug is selected from one of the following: injection, subcutaneous implant, tablet, powder, granule, capsule, oral liquid, and sustained-release formulation.

[0016] Beneficial effects

[0017] Through experimental verification, this invention synthesizes recombinant human secretory DDRGK1-Fc, which has good bone-promoting function and can be applied to the preparation of drugs for treating bone and joint-related diseases, promoting cartilage formation, and promoting intervertebral disc formation. This opens up new ideas for the treatment of bone and joint diseases and has obtained relatively solid scientific evidence. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the expression construct of recombinant human secretory DDRGK1-Fc.

[0019] Figure 2 A schematic diagram and gel image of the recombinant human secretory DDRGK1 and its expression construct using the HA signal peptide.

[0020] Figure 3 Gel images showing the purification of recombinant human secretory DDRGK1-Fc and recombinant human secretory DDRGK1 protein modified with HA signal peptide.

[0021] Figure 4 Figure A shows in vitro osteogenic data of secreted DDRGK1 and recombinant human secreted DDRGK1-Fc. Figure B shows the μCT analysis of mouse tibial defects 7 days after protein injection, with box 1 representing the secreted DDRGK1 protein group and box 2 representing the recombinant human secreted DDRGK1-Fc protein group. Figure C shows the statistical analysis of the number of trabeculae (Tb.N) after protein injection in mouse tibial defects. Figure D shows the statistical analysis of the bone volume fraction (BV / TV) after protein injection in mouse tibial defects.

[0022] Figure 5 To compare the osteogenic effects of secreted DDRGK1 protein, secreted DDRGK1-Fc protein and intracellular DDRGK1 protein, (A) shows the observation of extracellular matrix calcium deposition using 1% Alizarin Red S solution, and (B) shows the percentage of Alizarin Red staining area. Detailed Implementation

[0023] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0024] Example 1

[0025] (1) Experimental materials

[0026] 1.1 Instruments: High-speed centrifuge (Beckman Avanti JXN-26), mammalian cell shaker (Yonglian Biotechnology Co., Ltd.), AKTA PURE (GE)

[0027] 1.2 Materials: Human DDRGK1 gene (synthesized by Genewiz Biotechnology), Expi293F cells (Thermo Fisher Scientific, A14527), pcDNA3.4-HHC plasmid (laboratory modified), size exclusion chromatography column (GE, Superdex 200 Increase 10 / 300 GL), Ni Smart Beads 6FF (Tiandi Renhe, SA036100), TEV protease (Reference: Zhou F., et al., Crystal structure of a bacterial homolog to human lysosomal transporter, Spinster. Science). Bulletin (2019), 64(18), pp. 1310-1317 DOI: 10.1016 / j.scib.2019.08.010), SPINX centrifuge filter (BaiSai), human bone marrow mesenchymal stem cells h-BMSC: (Shanghai Key Laboratory of Orthopedic Implants, Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine), C57 mice: (Shanghai Key Laboratory of Orthopedic Implants, Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine).

[0028] 1.3 Reagents: 293 cell culture medium (Yonglian Biotechnology Co., Ltd., UP0050), transfection reagent PEI MAX (Polyscience, 24765),

[0029] (2) Experimental methods

[0030] ① Construction of recombinant expression vector and expression of target protein

[0031] Recombinant human secretory DDRGK1-Fc: The human DDRGK1 gene (NCBI reference sequence: NM_023935.2) was synthesized by Genewiz Biotechnology after codon optimization. Figure 1As shown, PCR yielded a truncated human DDRGK1 cDNA fragment encoding amino acid residues deleted from positions 1-28 (corresponding protein sequence: AS AGQEPLHNEE LAGAGRVAQP GPLEPEEPRAGGRPRRRRDL GSRLQAQRRA QRVAWAEADE NEEEAVILAQ EEEGVEKPAE THLSGKIGAK KLRKLEEKQARKAQREAEEA EREERKRLES QREAEWKKEE ERLRLEEEQK EEEERKAREE QAQREHEEYL KLKEAFVVEEEGVGETMTEE QSQSFLTEFI NYIKQSKVVL LEDLASQVGL RTQDTINRIQ DLLAEGTITG VIDDRGKFIYITPEELAAVA NFIRQRGRVS IAELAQASNS LIAWGRESPA The fragment (QAPA) was cloned into a laboratory-modified pCDNA3.4 plasmid after being double-digested with AscI / NotI. A membrane signal peptide HA (amino acid sequence: MKTIIALSYIFCLVFAGRA) was attached to the N-terminus of the gene sequence, and an Fc fragment (amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK) was attached to the C-terminus. Figure 1 The TEV proteaserecognition site is located between the DDRGK1 and Fc sequences.

[0032] The secretory form of the HA signal peptide DDRGK1 was used instead: the human DDRGK1 gene (NCBI reference sequence: NM_023935.2) was synthesized by Genewiz Biotechnology after codon optimization. Figure 1As shown, PCR yielded a truncated human DDRGK1 cDNA fragment encoding amino acid residues deleted from positions 1-28 (corresponding protein sequence: AS AGQEPLHNEE LAGAGRVAQP GPLEPEEPRAGGRPRRRRDL GSRLQAQRRA QRVAWAEADE NEEEAVILAQ EEEGVEKPAE THLSGKIGAK KLRKLEEKQARKAQREAEEA EREERKRLES QREAEWKKEE ERLRLEEEQK EEEERKAREE QAQREHEEYL KLKEAFVVEEEGVGETMTEE QSQSFLTEFI NYIKQSKVVL LEDLASQVGL RTQDTINRIQ DLLAEGTITG VIDDRGKFIYITPEELAAVA NFIRQRGRVS IAELAQASNS LIAWGRESPA The fragment (QAPA) was cloned into a laboratory-modified pCDNA3.4 plasmid after being double-digested with AscI / NotI. A membrane signal peptide HA (amino acid sequence MKTIIALSYIFCLVFAGRA) was ligated to the N-terminus of the gene sequence, and a TEV protease recognition site and affinity purification tag HHHHHHHH were ligated to the C-terminus. Figure 2-4 The DDRGK1 mentioned refers to the secretory DDRGK1 that uses the HA signal peptide.

[0033] Figure 5The secretory DDRGK1 protein: The human DDRGK1 gene (NCBI reference sequence: NM_023935.2) was synthesized by Genewiz Biotechnology after codon optimization. PCR yielded a truncated human DDRGK1 cDNA fragment encoding amino acid residues deleted from positions 1-28 (corresponding protein sequence: AS AGQEPLHNEE LAGAGRVAQP GPLEPEEPRA GGRPRRRRDLGSRLQAQRRA QRVAWAEADE NEEEAVILAQ EEEGVEKPAE THLSGKIGAK KLRKLEEKQA RKAQREAEEAEREERKRLES QREAEWKKEE ERLRLEEEQK EEEERKAREE QAQREHEEYL KLKEAFVVEE EGVGETMTEEQSQSFLTEFI NYIKQSKVVL LEDLASQVGL RTQDTINRIQ DLLAEGTITG VIDDRGKFIY ITPEELAAVANFIRQRGRVS IAELAQASNS LIAWGRESPA The fragment (QAPA) was cloned into a laboratory-modified pCDNA3.4 plasmid after being digested with AscI / NotI. The N-terminus of the gene sequence was linked to the membrane signal peptide MKTIIALSYIFCLVFA, and the C-terminus was linked to the TEV restriction site ENLYFQG and the purification tag HHHHHHHH.

[0034] ② Protein purification

[0035] Expi293F cells were cultured at 37°C and 5% CO2 until the cell count reached 2.5 × 10⁻⁶ cells / mL. 6After reaching the target density, transient transfection can be performed. Transfect 1 mg of plasmid per 1 L of cells. Premix the expression plasmid and transfection reagent PEI MAX at a 1:3 ratio (w / w) in 100 mL of fresh culture medium, incubate for 30 minutes, and then add the mixture to 1 L of Expi293F cells for 72 hours. Collect the supernatant after 72 hours. Centrifuge at 1500g for 10 minutes to collect the supernatant, then centrifuge the resulting supernatant again at 5000g for 20 minutes to collect the supernatant. Load 2 mL of Ni Smart Beads 6FF purification medium into an empty purification column. First, wash the medium with 20 column volumes of deionized water to remove the 20% ethanol solution, then pre-equilibrate the purification medium with 20 column volumes of buffer (20 mM Hepes pH 8.0, 150 mM NaCl, 10% Glycerol). Then add the supernatant to the equilibrated Ni Smart Beads 6FF medium and flow it through the column by gravity at a flow rate of 0.5 mL / min. After all the supernatant has been extracted, wash away any non-specifically bound impurities adsorbed on the purification medium with 20 column volumes of buffer. Finally, resuspend the medium in a centrifuge tube with 3 column volumes of buffer, ensuring thorough resuspending and no residue. Add 0.4 mg of TEV protease and incubate overnight at 4°C on a rotary shaker for enzymatic digestion.

[0036] The overnight digested samples from the centrifuge tubes were reloaded into an empty purification column. The flow-through was collected, and the purification medium was slowly washed twice with one column volume of buffer. Finally, all the flow-through was collected. All digested samples were concentrated to 1 ml, filtered through a SPINX centrifugal filter, and then further purified using a size exclusion chromatography column (Superdex 200Increase 10 / 300GL, GE Healthcare), pre-mounted on an AKTA PURE instrument. The buffer solution was 20 mM Hepes pH 8.0, 150 mM NaCl. After SDS-PAGE validation, the protein was collected for subsequent experiments.

[0037] ③ Osteogenic differentiation

[0038] Extracellular secretory DDRGK1 and secretory DDRGK1-Fc proteins were dissolved in Hepes buffer (20 mM Hepes, pH 8.0 + 150 mM NaCl) at a working concentration of 600 ng / ml. Human bone marrow-derived mesenchymal stem cells were used as the research subject, with 5 × 10⁻⁶ cells / ml. 4 They were seeded into 48-well plates at a density of 10 cells / well and cultured for 24 hours, then prepared with 15% FBS, 10 -7The original medium (MEMα medium containing 10% FBS) was replaced with a low-glucose DMEM medium containing mM dexamethasone, 5mM β-glycerophosphate, and 50 μg / mL ascorbic acid, at a density of 500 μL per well. The medium was changed twice a week until day 21. Extracellular matrix calcium deposition was observed using 1% Alizarin Red S solution (Solarbio, Beijing, China), and the results are as follows: Figure 4 A.

[0039] Using mouse embryonic osteoblast line 3T3-E1 as the research subject, 5×10 4 They were seeded into 24-well plates at a density of 10 cells / well and cultured for 24 hours, then prepared with 15% FBS, 10 -7 The original medium (MEMα medium containing 10% FBS) was replaced with a low-glucose DMEM medium containing mM dexamethasone, 5mM β-glycerophosphate, and 50 μg / mL ascorbic acid, at a density of 500 μL per well. The medium was changed twice a week until day 21. Extracellular matrix calcium deposition was observed using 1% Alizarin Red S solution (Solarbio, Beijing, China). Figure 5 As shown.

[0040] A defect was created approximately 1 mm in diameter on the anteromedial aspect of the tibia of mice using a 1 mm drill. Proteins were injected locally every two days, with both X protein and BMP2 protein concentrations at 15 ng / µl, at a total injection volume of 20 µl, for a total of three injections. MicroCT scans were performed 7 days post-surgery. (B) MicroCT analysis of the control group, recombinant human secreted DDRGK1-Fc protein group (box 2), inactivated recombinant human secreted DDRGK1-Fc protein group, secreted DDRGK1 protein group (box 1), inactivated secreted DDRGK1 protein group, and BMP2 protein group. (C) Statistical analysis of trabecular bone number (Tb.N). (D) Statistical analysis of bone volume fraction (BV / TV). *p<0.05, **p<0.005. Results are as follows. Figure 4 B, C, and D.

[0041] (3) Experimental Results

[0042] Figure 2 This indicates that after secretory DDRGK1 was replaced with the HA signal peptide (box 3 at the top left), ddrgk secretion increased (box 2).

[0043] Figure 3 This indicates that the secretory DDRGK1 (box 1) has an added FC segment (box 2).

[0044] Figure 4Figure A shows that secreted DDRGK1 protein has osteogenic function (box 1), and recombinant human secreted DDRGK1-FC also has osteogenic function (box 2). Moreover, the osteogenic effect of recombinant human secreted DDRGK1FC is better than that of secreted DDRGK1. Figure 4 Figures B and D show that, in the bone volume fraction (BV / TV%) index, the recombinant human secreted DDRGK1 FC was 0.1884±0.03831, while that of secreted DDRGK1 was 0.1672±0.03095. The promoting effect of recombinant human secreted DDRGK1 FC exceeded that of secreted DDRGK1 by 12.67%. Figure 4 Figures B and C show that, in the TbN index of trabecular bone number, the FC of recombinant human secretory DDRGK1 was 5.786±0.8829, while that of secretory DDRGK1 was 5.584±1.106, with the promoting effect exceeding that of DDRGK1 by 3.6%.

[0045] Figure 5 (A) shows that secreted DDRGK1-Fc protein has the strongest osteogenic effect (box 1), followed by secreted DDRGK1 protein (box 2). Both are stronger than the osteogenic effect of intracellular DDRGK1 protein, i.e., the OE-DDRGK1 group (box 3). (B) shows that the osteogenic effect of secreted DDRGK1 protein in the PBS group was 44.07±6.629%, while that in the O / EC group was 69.00±2.234. The osteogenic effect of secreted DDRGK1 protein exceeded that of intracellular DDRGK1 by 56.5%. The osteogenic effect of O / EC group was 78.43±2.603, which exceeded that of intracellular DDRGK1 by 77.9% and that of secreted DDRGK1 by 13.7%.

Claims

1. A recombinant human secreted DDRGK1-Fc, characterized in that, The DDRGK1-Fc consists of, from N-terminal to C-terminal, a signal peptide HA shown in SEQ ID NO. 1, a truncated human DDRGK1 shown in SEQ ID NO. 3, a TEV enzyme cutting site with an amino acid sequence of ENLYFQG, and an Fc fragment shown in SEQ ID NO.

2.

2. Use of the recombinant human secretory DDRGK1-Fc of claim 1 in the preparation of a medicament for treating bone fracture or bone defect.

3. A medicament comprising the recombinant human secretory DDRGK1-Fc of claim 1.

4. The medicament according to claim 3, characterized in that, The dosage form of the medicament is selected from one of injection solution, subcutaneous implant, tablet, powder, granule, capsule, oral solution, and sustained release agent.

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